Method for regulating neuron development and maintenance

ABSTRACT

The present invention relates to a method for regulating neuron development/maintenance and/or regeneration in a nervous system of a mammal and to pharmaceutical compositions comprising leukaemia inhibitory factor useful for same.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation U.S. application Ser. No.10/422,552, filed Apr. 24, 2003, which is a continuation of applicationSer. No. 09/568,003, filed May 10, 2000, now abandoned, which is acontinuation of application Ser. No. 08/410,402, filed Mar. 27, 1995,now U.S. Pat. No. 6,177,402, which is a File Wrapper Continuation ofapplication Ser. No. 07/923,939, filed Mar. 20, 1991, now abandoned.

The present invention relates to a method for regulating neurondevelopment, maintenance and regeneration in the central and peripheralnervous systems of a mammal and to pharmaceutical compositionscomprising leukaemia inhibitory factor useful for same. The presentinvention is particularly useful in the treatment of developmental andcerebral anomalies and neuropathies in mammals and in particular humans.

Leukaemia Inhibitory Factor (hereinafter referred to as “LIF”) is aprotein that has been purified, cloned and produced in large quantitiesin purified recombinant form from both Eschericia coli and yeast cells(International Patent Application PCT/AU88/00093). LIF was originallyisolated on the basis its capacity to induce differentiation andsuppression of the murine myeloid leukaemic cell line, M1. LIF has noapparent proliferative effect on normal haematopoietic cells althoughLIF receptors have been detected on cells of the monocyte-macrophagelineage.

The present invention arose in part from an investigation of the effectsof LIF on cells of the neural crest. The neural crest is a population ofprecursor cells which arises from the dorsal lip of the neural tubeduring embryogenesis and migrates through the embryo along a complexseries of pathways. After migration the crest cells give rise to a greatvariety of cell types including the neurons and Schwann cells of thesensory and autonomic ganglia, the enteric nervous system, adrenalmedulla, melanocytes of the skin and facial mesenchyme. When studied atthe population level, the crest appears to be a multipotent collectionof stem cells. The extensive transplantation experiments of Le Douarinand colleagues, whereby quail neural crest were grafted into chickembryos, showed that the developmental fate of the crest cells wasdetermined by the location of this graft in the chick embryo (1). Thisnot only indicated that the full developmental repertoire of the crestis contained in the different subpopulations of grafted crest cells, butalso that environmental factors play a major role in the finaldifferentiated phenotype of the cells.

In the last decade it has become increasingly clear that the neuralcrest contains subpopulations of cells which are already committed toparticular developmental pathways (2,3). However, it is also clear thatthe differentiation of these cells is determined by environmentalfactors.

A number of soluble trophic factors have been shown to act as survivalagents for neural crest derived neurons, but none of these have beenshown to act directly on the neuronal precursor cells within the neuralcrest. These factors include nerve growth factor (NGF; 4), brain-derivedneurotrophic factor (BDNF; 5), ciliary neurotrophic factor (CNTF; 6) andthe fibroblast growth factors (FGF's; see 5).

In work leading up to the present invention, experiments were conductedto locate an agent having direct effect on the precursor populations ofthe neural crest. In accordance with the present invention, it has beensurprisingly discovered that neural crest cells differentiate into fullymature neurons in the presence of LIF. This effect is titratable andoccurs in the absence of proliferation of neuronal precursor cells.Furthermore, the effect of LIF on the differentiation of neural crestcells into neurons extends to the stimulation of the differentiation ofprecursor cells in embryonic dorsal root ganglia into mature sensoryneurons.

Accordingly, one aspect of the present invention contemplates a methodfor regulating neuron development and/or maintenance and/or regenerationin a mammal comprising administering to said mammal an effective amountof leukaemia inhibitory factor (LIF) for a time and under conditionssufficient to permit the differentiation and/or maintenance and/orregeneration of neural precursor cells into neurons.

Another aspect of the present invention relates to a method forenhancing and/or stimulating and/or maintaining and/or regenerating theformation and/or survival of neurons in the central nervous system of amammal which comprises administering to said mammal an effective amountof LIF for a time and under conditions sufficient to effect an increasein and/or to maintain the number of neurons in the central nervoussystem.

In one embodiment, the LIF enhances, stimulates, maintains (i.e.promotes survival) and/or regenerates immature neurons.

Yet another aspect of the present invention relates to a method forenhancing, stimulating and/or maintaining the formation and/or survivalof sensory neurons, for example sensory neurons, of the peripheralnervous system of a mammal which comprises administering to said mammalan effective amount of LIF for a time and under conditions sufficient toeffect an increase in and/or to maintain the number of neurons in theperipheral nervous system.

By “LIF” as used herein is meant to include naturally occurring,recombinant and synthetic LIF comprising the naturally occurring aminoacid sequence or any single or multiple amino acid substitutions,deletions and/or additions therein including single or multiplesubstitutions, deletions and/or additions to any molecules associatedwith LIF such as carbohydrate, lipid and/or peptide moieties.Accordingly, the term “LIF” as used herein contemplates naturallyoccurring LIF and LIF-like polypeptides which include mutants,derivatives, homologues and analogues of LIF. Regardless of the LIFmolecule used, however, the only requirement is that it can assist inregulating neuron development and/or maintenance and/or regeneration ina mammal. In a preferred embodiment the mammal is human and the LIF isof human origin or from a different mammal but which still has activityin a human. Hence, the source of LIF and the mammal to be treated may behomologous, i.e. from the same mammal or may be heterologous, i.e. froma different mammal. In some circumstances, the mammal to be treated mayitself be used to isolate the LIF for use in the method of the presentinvention.

By “regulating neuron development, maintenance and regeneration” as usedherein is meant to include stimulating, enhancing and/or maintaining theformation and/or survival of neurons in the central and peripheralnervous systems of a mammal. It also includes the ability of said factorto assist the regeneration of properties associated with neuronalfunction following damage caused by disease or trauma. It is alsoincludes stimulating, enhancing, maintaining and/or regenerating thoseproperties associated with neurons such as, but not limited to,neurotronsmitter type, receptor type and other features associated withthis phenotype. In articular, LIF has been shown herein to induce,stimulate, enhance, maintain and/or regenerate the differentiation ofneural crest cells into fully mature neurons. This effect is titratableand occurs in the absence of proliferation of neuronal precursor cells.The effect of LIF also extends to the stimulation of the differentiationof precursor cells in embryonic dorsal root ganglia (DRG) into maturesensory neurons. The sensory neurons of the peripheral nervous systemare derived from precursor cells in the embryonic neural crest. Aftercrest migration, these precursor cells aggregate into the DRG and thendifferentiate into mature sensory neurons. The survival of sensoryneurons has been shown to be dependent on two characterised growthfactors, nerve growth factor (NGF) and brain derived neurotrophic factor(BDNF) and other undefined factors at critical stages duringdevelopment. However, nothing is known about the identity of factorswhich might stimulate the differentiation of the sensory precursorcells. It was, therefore, surprisingly found in accordance with thepresent invention that LIF stimulated the differentiation of precursorcells in the embryonic DRG into mature sensory neurons and that LIFacted as a survival factor for these neurons throughout embryogenesisand into postnatal life.

LIF also affects the central nervous system. The early steps in thedevelopment of the central nervous system from the embryonic precursorcells of the neural tube involves expansion of the precursor populationand differentiation of these cells into mature neurons and glia. Thisphase is followed by a selective survival of neurons which haveappropriately innervated the correct targets and is believed to be basedon the limited availability of survival factors which are produced bythe target cells.

It has been recently shown (9) that the fibroblast growth factors areinvolved in the expansion and differentiation phases of development ofthe embryonic brain and in addition it has also been shown that FGF canact as a survival agent for mature neurons. Work from Barde (5)indicates that the survival of a subset of CNS neurons, the retinalganglion cells, is dependent on BDNF. However, little is known aboutother factors which are operative in the development of the embryonicbrain and spinal cord.

Accordingly, it has now been surprisingly found that LIF acts as adifferentiation/survival and/or regenerating agent for spinal cordneurons and enhances, stimulates and/or promotes spinal cord developmentand promotes neurite extension.

This method is particularly applicable to regulating spinal corddevelopment and in treating a disease, injury and/or an abnormality to anervous system. For example, the method of the present invention can beused in relation to the central and/or peripheral nervous system totreat one or more of Cerebral Palsy, trauma induced paralysis, vascularischaemia associated with stroke, neuronal tumours, motorneuronedisease, Parkinson's disease, Huntington's disease, Alzheimer's disease,multiple sclerosis, peripheral neuropathies associated with diabetes,heavy metal or alcohol toxicity, renal failure and/or infectiousdiseases such as herpes, rubella, measles, chicken pox, HIV and/orHTLV-1.

Another aspect of the invention relates to a method for enhancing,stimulating, maintaining and/or regenerating spinal cord development andspinal cord neuron number which comprises administering to said mammalan effective amount of LIF for a time and under conditions sufficient toeffect an increase in spinal cord neuron number and spinal corddevelopment.

Yet another aspect relates to a method of enhancing, stimulating,maintaining and/or regenerating neurite extension from spinal cord andother central nervous system neurons and further relates to the centralnervous system other than the spinal cord.

Still yet another aspect of the invention contemplates a method oftreatment of disease and injury in both the central and peripheralnervous systems in a mammal, said disease or injury including but notlimited to one or more of Cerebral Palsy, trauma induced paralysis,vascular ischaemia associated with stroke, neuronal tumours,motorneurone disease, Parkinson's disease, Huntington's disease,Alzheimer's disease, multiple sclerosis and peripheral neuropathiesassociated with diabetes, heavy metal or alcohol toxicity, renal failureand/or infectious diseases such as herpes, rubella, measles, chickenpox, HIV and/or HTLV-1 which comprises administering to said mammal aneffective amount of LIF for a time and under conditions sufficient toameliorate the disease or injury.

In all such methods of the present invention, the enhancing,stimulating, maintaining and/or regenerating of neurons is referred toas “regulating neuron development”. Furthermore, use of the term “LIF”includes LIF-like polypeptides and derivatives thereof as discussedabove.

The effective amount of LIF used in accordance with the presentinvention will be that required to regulate the neurons and willgenerally be from about 0.01 to about 10,000 microgram (μg) per kilogram(kg) of body weight and preferably 0.1 to 10,000 μg/kg and mostpreferably 1 to 1000 μg/kg of body weight. However, depending on suchfactors as the disease treated, the treatment and the patient, more orless LIF may be used while still being within the scope of the presentinvention. Furthermore, it may be convenience to determine the effectiveamount of LIF in Units/ml or Units/kg. The definition of a Unit of LIFactivity can be found in PCT/AU88/00093. For example, and not by way oflimitation LIF may be used from 10 to 10⁸ U/ml. Administration may beper hour, per day, per week or per month or may be a singleadministration. Administration may also need to be continuous infusion.

In accordance with the present invention, LIF may be administered aloneor in combination with one or more other neuron stimulating factors suchas, but not limited to, FGF, CNTF and/or BDNF and/or other neurotrophicfactors. In “combination” means either the simultaneous addition of LIFand the one or more other factors in the same composition or thesequential addition of the LIF and one or more other factors where afirst factor is given followed by a second factor. The exact order ofaddition and time between additions is best determined by the practicingphysician and may depend on the patient and/or the treatment required.

Accordingly, the one or more other neuron stimulating factors may begiven by simultaneous or sequential administration with LIF. Theeffective amount of other neuron stimulating factors will be from about0.01 to about 10,000 μg/kg body weight, preferably 0.1 to 10,000 μg/kgand most preferably 1 to 1,000 μg/kg body weight. Again, administrationmay be a single dose or repeated per hour, per day, per week or permonth. Administration may also be continuous infusion.

The route of administration is preferably by intramuscular orintravenous injection or using gene therapy although other routes ofadministration are possible such as by infusion, drip, intracerebralinjection and/or implants.

Another aspect of this invention relates to administration of LIF totarget tissue, or the precise location of the nerve so as to facilitateuptake by retrograde transport as outlined in Example 5.

The present invention is also directed to a pharmaceutical compositioncomprising LIF and one or more neuron stimulating factors and one ormore pharmaceutically acceptable carriers and/or diluents. Such acomposition is useful for regulating neuron development and/ormaintenance in a mammal such as in enhancing, stimulating, maintainingand/or regenerating the formation and survival of neurons in theperipheral nervous system and/or enhancing, stimulating, maintainingand/or regenerating the formation and survival of sensory neurons in thecentral nervous system and/or enhancing, stimulating and/or maintainingthe formation and survival of spinal cord neurons and/or spinal corddevelopment.

Preferably, the composition is suitable for administration into a human.In accordance with the present invention, the LIF used in thecomposition is as previously herein defined and includes, for example,LIF-like polypeptides and mutants, derivatives, homologues and/oranalogues of LIF. The LIF and other neuron stimulating molecules and/orneurotrophic factors may be the same or different in terms of theirmammalian source and whether they are naturally occurring, recombinantor synthetic. As with the method, the mammalian source of the LIF andother neuron stimulating factor may be homologous or heterologous to themammal being treated. The compositions of the present invention are alsouseful in treating the diseases, injuries and/or abnormalities of anervous system as previously described.

The preparation of pharmaceutical compositions is well known in the artand reference can conveniently be made to Remington's PharmaceuticalSciences, 16th ed., 1980, Mach Publishing Co., Edited by Osol et al.

Another aspect of the present invention is directed to the use of LIFincluding its derivatives for the manufacture of a medicament forenhancing, stimulating, maintaining and/or regenerating the formationand/or survival of neurons in the peripheral nervous system and/orenhancing, stimulating maintaining and/or regenerating the formationand/or survival of neurons in the central nervous system and/orenhancing, stimulating, maintaining and/or regenerating the formationand/or survival of spinal cord neurons and/or spinal cord development ina mammal. Preferably, the mammal is a human and the LIF used is ashereinbefore defined. The use in accordance with the present inventionmay also include the use of one or more other neuron stimulating factorssuch as FGF, CNTF and/or BNDF.

The present invention is further described by reference to the followingnon-limiting figures and examples.

In the Figures:

FIG. 1 shows the effect of LIF on neuron numbers in neural crestcultures. Neural crest cells were incubated in medium alone or in thepresence of LIF for 6 days, Nissl stained (8), and neurons were countedusing bright field microscopy. In the “− tube” experiment, neural tubeswere removed after 24 h and LIF was added to the cultures. Neuronnumbers could not be accurately counted at later times because of denseclustering of neurons in LIF cultures. Numbers are the mean and standarddeviation, n=6. *P<0.005, **P<0.05; t-test.

FIG. 2 is a photographic representation showing the phenotype of neuronsin neural crest cultures. Neural crest cultures were incubated for 13days in the presence (b,d,e,f,g) or absence of LIF (a,c).Photomicrographs shown are: a, b, bright field views of Nissl stained(8) cultures; c,d, fluorescence views of cultures stained forneurofilament. e, bright field view of LIF treated culture stained forCGRP. f, bright field view of LIF treated culture stained for tyrosinehydroxylase. g, fluorescence view of same field as in (f). bar=200 μm(a,b), 50 μm (c,d,e,f,g).

FIG. 3 is a photographic representation showing. ³H-thymidineincorporation into neural crest cultures. ³H-thymidine (0.03 μC/ml) andLIF (10⁴ U/ml) were added after 4 days of culture and incubationcontinued for another 9 days, following which cultures were stained forneurofilament and autoradiographed (9). a, bright field photomicrographof culture; b, fluorescence view of same field. bar=50 μm.

FIG. 4 contains graphical representations showing: A the effect of LIFon neuron numbers in cultures of E12-P2 DRG. DRG cells were plated inMonomed medium, 10% FBS (control, black bars) or +LIF (10⁴ U/ml, hatchedbars) and neuron numbers determined after 5 days (E12) or 2 days (othercultures) as described in Example 1. Numbers of neurons and cellsinitially plated are given in Example 1; B, limit dilution analysis ofneuron survival in P2 DRG cultures. Cells (70% neurons, of which 75%plated after 2 hrs) were plated at the indicated number (120wells/dilution) in the presence (diamonds) or absence (squares) or 10²U/ml LIF and wells with live neurons were counted after 2 days. A linearrelationship exists between input cells number and the log of the %negative wells (R=0.992), indicating that the effect of LIF on neuronsurvival obeys zero order (single hit) kinetics (11); C, dose-responserelationship of neurons to LIF concentration in P2 DRG cultures. P2 DRGcells (200/well) were plated with the indicated concentration of LIF andneurons were counted after 2 days. Mean and standard deviation are shownin A and C. n=6

FIG. 5 is a photographic representation showing photomicrographs ofexplants of E10 spinal cords cultured in the presence of LIF in 24 wellplates at D7 in vitro to display process outgrowth. Shown are cultureswith a) no LIF b) LIF (Bar=100 um).

FIGS. 6 a,b are photographic representations showing the morphology ofcultures arising from LIF stimulated spinal cord cells. Cells insuspension were plated as described in Example 1 and incubated in 96well plates for 5 days. Shown are phase-contrast photographs of cellsincubated with a) no LIF b), LIF (Bar=100 um).

FIGS. 6 c,d are photographic representations showing cultures of spinalcord precursors stained for neurofilament antibody. Cells were plated asdescribed in Example 1 and incubated in HL-A plates for 5 days prior tofixation and staining. Shown are fluorescence photomicrographs of cellsincubated with c) no LIF d) LIF (Bar=100 um).

FIG. 7 is a graphical representation showing the effect of LIF onprocess outgrowth. E10 spinal cord precursors (5×10⁴) were plated in thepresence or absence of LIF (10⁴ U/ml) in 96 well plates for 5 days, asdescribed in materials and methods. The number of processes emanatingfrom each discrete clump of cells to aggregate, was quantitated. Thefrequency of clumps with a given number of processes was determined. Thefrequencies for every 5 increments of processes/clump (e.g. 0-4, 5-9)were aggregated and expressed as a % of the total number of clumps perwell. These frequencies were averaged for six wells in both LIF treatedand control cultures and the means and standard deviations are expressedin the graph.

FIG. 8 is a graphical representation showing binding of ¹²⁵I-LIF tosensory neurons from dorsal root ganglion. Binding (solid bars) isalmost exclusively restricted to neurons as shown in (B) and notaccessory cells (A). Virtually all the binding is inhibitible by coldLIF (hatched bars) indicating that binding is specific.

FIG. 9 is a graphical representation showing retrograde Transport of¹²⁵I-LIF by Sciatic Nerve in the adult mouse. Significant accumulationis found in L3, L4, L5 dorsal root ganglia when injections are made intothe foot pad (solid bars).

FIG. 10 is a graphical representation showing retrograde Transport of¹²⁵I-LIF to the Sensory Ganglia in the newborn mouse. Significantaccumulation of label again centred on L4 although this occurred withboth foot and muscle injection. There was some uptake by more rostralsensory ganglia when injections were made intra-muscularly.

FIG. 11 is a photographical representation showing an autoradiograph ofsection through L4 dorsal root ganglia showing accumulation of silvergrains over a small population of neurons (a) ×400. Note that only theneurons label, not the Schwann cells (accessory cells) (b) ×1000.Sections stained with haematoxylin and eosin.

FIG. 12 is a photographic representation showing the distribution ofgrain counts in sections of L4 dorsal root ganglia after retrogradelabelling with ¹²⁵I-LIF. Note only a small proportion (5-10%) havesignificantly labelling.

FIG. 13 is a graphical representation showing spinal cord cellssurviving in vitro with and without LIF over time.

EXAMPLE 1 Materials and Methods

Preparation of Neural Crest Cells

CBA mouse embryos at embryonic day 9 (E9) were removed from the uterusand placed in a petri dish containing Hepes buffered Eagles Medium (HEM)with 1% (v/v) fetal bovine serum (FBS). The head and tail were removedusing 26 gauge syringe needles with the aid of a dissecting microscopeleaving a trunk segment with 8-12 somites each side of the neural tube.These trunk segments were placed in a fresh petri dish in HEM 1% (v/v)FBS and the somites and surrounding tissue were carefully removed fromthe neural tube using 26 gauge needles. One or two tubes were thenplaced in each well of a 24 well plate (Linbro) which had beenpreviously coated with fibronectin (5 ug/ml). Dulbecco's modifiedEagles' medium (DME) with 10% (v/v) FBS was then carefully run down theside of each well, so that it almost covered the bottom of the well.This enabled the neural tubes to associate with the fibronectinsubstratum and adhere. In particular experiments, the tubes werecarefully removed after 24 hrs, leaving a layer of migratory neuralcrest cells. In other experiments the tubes were left in the wells so asnot to disturb any of the integrated crest cells. Monomed medium(Commonwealth Serum Laboratories, Parkville, Victoria, Australia) with10% (v/v) FBS and the specified growth factors was added to 1 ml to allcultures after 24 hrs. Cultures were incubated at 37° C. in 5% CO₂/95%air.

Removal of Dorsal Root Ganglia (DRG)

Two day old neonatal mice were decapitated under aseptic conditions andplaced into sterile petri dishes. The trunk was washed with a solutionof 70% (v/v) ethanol in distilled water. A vertical incision through theskin was made using a sterile pair of 45° angle bladed scissors. Allinstruments used had previously been soaked for one hour before use in asolution of 70% (v/v) ethanol in distilled water.

A fine pair of iris scissors was used to make an incision through thedorsal aspect of the spinal column, which enabled the spinal cord to beremoved using a pair of curved watchmaker forceps. This exposed thedorsal root ganglia and facilitated their removal. A sterile piece ofgauze was used to swab the area around the ganglia so as to adsorb anyblood and tissue fluid that obscured the view of the ganglia. Then usinga pair of straight very fine tipped forceps each ganglia was carefullyremoved free of surrounding spinal tissue and placed into a petri dishin a small volume of N-2 hydroxyethylpiperasine-N-20 ethanesulfonic acid(HEPES) and buffered Eagles minimal essential medium (HEM).Approximately twenty ganglia were removed from each mouse.

DRG Cultures

The DRG dissected free of surrounding spinal tissue and placed in HEM,were finely chopped, then incubated in HEM, 0.025% (w/v) trypsin, 0.001%(w/v) DNase at 37° C. (12 min for E12, 20 min for E15 and 30 min for E19and P2). FBS was added to 20% (v/v), the cells were centrifuged at 300 gfor 5 min, washed twice in HEM, 0.01 (w/v) DNAse and triturated through18-25 gauge needles to obtain a single cell suspension. DRG cells wereplated onto fibronectin coated (15 μg/ml) wells of HL-A plates (Nunc,II) at previously optimised cell numbers (3500 cells at E12, 1000 atE15, and 200 at E19 and P2). Two hrs after plating, no mature neuronswere observed in the E12 cultures and an average of 110, 120 and 100neurons had were present in the E15, E19 and P2 cultures, respectively.Cultures from E12 were fixed and stained for neurofilament after 5 daysand neurofilament positive neurons counted using fluorescencemicroscopy. Neurons in later embryonic cultures (large, phase bright,round cells) were counted after 2 days.

Immunohistochemistry

For staining with particular antibodies neural tubes were plated ontoglass coverslips in 24 well plates or onto plastic microscopic slides(Nunc, 2 chamber slides). For staining with antibodies to neurofilament,the cells were first fixed in methanol at −20° C., washed 3 times in PBSand incubated for 30 min with an anti-neurofilament antibody (Chemicon)diluted 1:10 in HEM, 1% (v/v) FBS. The wells were then washed andincubated with a fluorescein isothiocyanate conjugated FITC sheepanti-rabbit antibody (Silenus) diluted 1:50 in PBS 1% (v/v) FBS, washedin PBS then in water, air dried and the cover slips mounted in 2.6% 1,4Diazobicyclo (2,2,2) octane in PBS/glycerol (1:9) Merck, Aust. To stainfor calcitonin gene related peptide (CGRP), cultures were fixed inparaformaldehyde (PFA), cleared with DMSO, washed with PBS, incubatedwith a rabbit anti-rat α-CGRP antibody (obtained from Dr G Olley, MonashUniversity, Aust., and which shows 7% binding to B-CGRP, <0.01% bindingto calcitonin, and negligible binding to substance P, Neurokinin A orEnkephalines by radio-immunoassay), washed and antibody binding detectedusing biotin conjugated second antibodies, a biotin-avidin-horseradishperoxidase complex (Vectastain ABC) and development withdiamino-benzidine. To stain for tyrosine hydroxylase or choline acetyltransferase (ChAT), cultures were fixed in PFA (and picric acid forChAT) incubated with a rabbit anti-tyrosine hydroxylase antibody (EugeneTech. USA) or a rat antiserum prepared against porcine ChAT (whichrecognises ChAT in the PNS (12), respectively and binding was detectedwith fluoresceinated second antibodies.

Thymidine Incorporation Experiments

To look for proliferating neural crest cells, ³H-thymidine (Amersham,specific activity 103 Ci/mmol) was added to the cultures at 0.1 or 0.03uCi/ml at the same time as growth factors were added or at correspondingtimes in control cultures. After 13 days some cultures were fixed inmethanol, stained for neurofilament as described above and then dippedin Kodak NT-B2 emulsion and exposed for 2 weeks at 4° C. and thendeveloped.

Isolation of Spinal Cord Cells

Embryos were obtained from embryonic day 10 (E10) mice. The heads wereremoved and the caudal part of the neural tube, or embryonic spinalcord, which forms a closed tube by E10, was removed together with thesurrounding somites from the remainder of the embryo. The section of thecord used in all experiments extended from the otic vesicle to justbelow the developing hind limb. This tissue was subsequently incubatedin Dispase II (Boehringer) in HEPES-buffered Eagle's medium (HEM) for 15minutes at 4° C. and for 6 minutes at 37° C. The tissue was thentransferred to HEM containing 1.0 (w/v) fetal bovine serum (FBS) and0.001% (w/v) DNase and the spinal cord was dissected free of thesurrounding ectoderm, somites and meninges, using the tissue platecreated by Dispase incubation essentially as described previously forthe preparation of the mesencephalic and telencephalic regions of theneural tube (9). Inspection at this stage revealed clean spinal cordsfree of contaminating mesoderm. These cords were plated directly forexplant cultures into 24 well plates (Linbro). For preparation ofdissociated cell suspensions, the spinal cords were then incubated at37° C. in Hank's with 0.02% (w/v) EDTA, 10 mM Hepes, 0.025% (w/v)trypsin and 0.001% (w/v) DNase pH7.6 for 12 minutes. The reaction wasstopped by the addition of FBS, the cells were washed in Ca²+/Mg²+ freeHank's and single cells were prepared by gently triturating thesuspension. An average of 1.5×10⁵ cells were obtained from thedissection of each embryo.

Primary Culture of Dissociated Spinal Cord Cells

Spinal cord cells (5×10⁴) were plated into 96 well plates (Linbro)coated with fibronectin (50 μg/ml) in Monomed medium and 0.05% FBS in afinal volume of 100 μl. Except where otherwise stated, LIF (murinerecombinant, specific activity+10⁸ U/mg) was used at a concentration of10⁴ Units/ml. Assays were normally performed over 5 days after which thecultures began to deteriorate. Cell counts were performed afterharvesting the cells with trypsin and triturating them. Processoutgrowth was quantitated at day 5 by scoring the number of processesemanating from each discrete clump of cells. Numbers in all cases arethe mean and standard deviation of six determinations. Cells were alsoplated onto confluent, irradiated (4000 Rad) monolayers of Balb/c-3T3cells on glass microscope slides in 24 well plates in Monomed medium and0.05% (v/v) FBS, at a density of 5×10³ cells/well. At the specifiedperiods of time, coverslips were fixed and stained for neurofilament asdescribed below and the number of positively stained cells per slide wasquantitated.

Purification of Radioiodination of LIF and FCF:

Recombinant LIF was produced in E. coli as a non-glycosylated protein.The purified species electrophoresed with an apparent molecular weightof 20,000 and an iso-electric point of greater than 9.0. Iodination ofLIF was performed by the iodine monochloride method as previouslydescribed (18). Briefly, 6 μl of a 1 mg/ml solution of LIF in 40% (v/v)acetonitrile, 0.1% (v/v) trifluoroacetic acid and 0.02% (v/v) Tween 20was iodinated by addition of 1 mCi Na¹²⁵I (New England Nuclear,North-Ryde, NSW, Australia), 40 μl of 200 mM sodium phosphate, 0.02%(v/v) Tween 20 at pH 7.4 (PBS) and, while vortex mixing, 5 μl of 200 μlof IC1 in 2M NaCl. After 1 min at room temperature radioiodinated LIF(¹²⁵I-LIF) was separated from unincorporated ¹²⁵I by sequential gelfiltration and cation-exchange chromatography. ¹²⁵I-LIF produced in thismanner retained full biological activity, was more than 99% precipitablewith cold 20% (w/v) trichloracetic acid and greater than 90% of theradioactivity was capable of binding specifically to M1 cells (17). Thespecific radioactivity was 1.1×10⁶ cpm/mole, as determined byself-displacement analyses. I¹²⁵ labelled aFGF was obtained as a giftfrom the RCC (Amersham). The specific activity of aFGF was 800 Ci/mM.

Binding Experiments and Autoradiography:

Dorsal root ganglion cells were obtained from postnatal day 2 mice asdescribed above and were cultured in 8 well microscope slides (Nunc) inmonomed medium containing 10% (v/v) FCS, but no added growth factorsovernight in a humidified incubator at 37° C. The slides were incubatedon ice for 2 hours in 200 μl of Hepes buffered ROMI-1640 medium,supplemented with 10% (v/v) FCS, 20 μl of with or without 10 μg/ml ofunlabelled LIF and from 5×10⁴ cpm of ¹²⁵I-LIF in 20 μl of DME, 10% (v/v)FCS. The cells were washed three times with 300 μl of PBS and fixed in10% (v/v) formalin in PBS for 2 hours and then rinsed in water. Slideswere dipped in Kodak NTB2 photographic emulsion at 42 C in a darkroomand allowed to dry. Slides were then sealed in a light-proof boxcontaining Drierite and exposed for 2-8 weeks at 4 C. Prior todevelopment, slides were warmed to room temperature and developed for 3minutes in Kodak D19 developer (40 g/500 ml of water), washed for 1minute in 0.5% (v/v) acetic acid in water and fixed in Agfa G333c X-rayfixer for 3 minutes. Slides with cytospin preparations were stained in5% (v/v) filter Giemsa in water for 10 minutes, dried and mounted inDepex. DePeX (BDH, Melbourne, Australia). Autoradiographs were examinedat ×400, ×650, or ×1000 magnification and where necessary, grain countswere performed on 100 consecutive cells of each type and backgroundcounts, in general between 0-3 grains, were subtracted.

Retrograde Labelling Experiments:

The sciatic nerves of newborn and adult Balb/C mice were ligated on oneside using 6-0 prolene monofilament (Ethicon). The radioactive proteinswere then injected either into the skin of the foot or intramuscularlyinto the centre of the gastronemus muscle. After appropriate times theanimals were killed by ether overdose and the sciative nerves disected.The nerves were cut at the ligation and 2 mm pieces were takenimmediately proximal and distal to the cut and counted directly.

Newborn and adult mice were injected in the footpad and kept for 16hours. Ganglia from T13 to S1 were removed under a disecting microscopeand the radioactivity estimated in the whole ganglion in a gammacounter. Selected ganglia or spinal cords with attached ganglia weredissected from the animals and fixed in 4% paraformaldehyde in PBS priorto being embedded in emulsion. Autoradiographs were developed 3-4 weekslater and ganglia and spinal cord examined for labelled cells.

EXAMPLE 2 Effect of LIF on Neural Crest Cells and Sensory Neurons

To examine the effect of LIF on neural crest cells, neural tubes weredissected from the cervical and thoracic region of E9 CBA mice, platedonto fibronectin coated wells and neural crest cells were allowed tomigrate onto the substratum for 24 hr, at which time the neural tubeswere either removed or left in place and LIF was added to the cultures.After two days, round cells with uni- or bi-polar processes, resemblingsensory neurons, appeared in the cultures. In the LIF treated culturesthere were approximately 12 fold more of these cells than in controls by6 days (FIG. 1) and they formed large clusters which increased in sizeup to 14 days (FIG. 2 b). This was not dependent on the presence of theneural tube during the culture period although, in their absence, theabsolute number of neuron-like cells was smaller (FIG. 1). Theseneuron-like cells stained positively with the Nissl stain (8), (FIGS. 2a and b) and for 150 kD neurofilament (13), (FIGS. 2 c and d).

This staining showed fine processes emanating from the clusters (FIG. 2d), confirming their neuronal phenotype. While the effect of LIF wasgreatest when added at day 1, it was still apparent when added at day 7.

To characterise the phenotype of neurons generated in these cultures,they were stained for the expression of markers found in sensory andautonomic neurons. All the neurons in both LIF treated and controlcultures contained immunoreactivity for CGRP (FIG. 2 e), the most widelyexpressed peptide found in mammalian sensory neurons (14,15). Limiteddevelopmental studies suggest that this peptide is expressed quiteearly, at least in the chick (18). Immunoreactivity for substance P, apeptide also found in mammalian sensory neurons (14,15), but only insignificant level postnatally (17), was also detected in a smallproportion of processes in both LIF treated and control cultures. Asmall proportion (1-2%) of these neurons (both LIF treated and control)had tyrosine hydroxylase activity, a marker for catecholaminergic cells(FIG. 2 f). However, none of the cells showed any immunoreactivity forCHAT, a marker for cholinergic cells.

These immunohistochemical findings, as well as the morphology of theneurons, suggest that they are in the sensory lineage. Previous work inaves has shown that at least a proportion of sensory neurons arise fromnon-dividing precursors in the neural crest (1-2). To investigatewhether the neurons in the LIF treated cultures also arose fromnon-dividing precursors, ³H-thymidine was added to the culturesconcomitantly with LIF at days 1, 4 and 7 of culture. Autoradiographicanalysis at day 13 showed that less than 0.2% of the neurons (2 in 1100neurons counted) which arose in the LIF cultures incorporated³H-thymidine (FIG. 3) irrespective of time of addition. Theseobservations show that the increase in neuron numbers does not resultfrom stimulation of precursor division. Most of the non-neuronal cellsin these cultures were labelled with ³H-thymidine (FIG. 3) but thepresence of LIF made no significant difference to the total proportionof labelled cells: when LIF was added on day 1, 80+/−18% of the cellswere labelled compared to 78+/−12% on control cultures, whereas at day7, 70+/−1% AND 70+/−6% of all cells were labelled in the presence andabsence of LIF, respectively [n=3].

As LIF stimulates an increase in sensory-like neurons in neural crestcultures, it was anticipated to have similar activity on early embryonicDRG cultures. Thus, single cell suspensions were made from E12 DRG,which contain a subpopulation of small, probably immature neurons aswell as neuronal precursors (18) and were plated into wells of HL-Aplates in the presence or absence of LIF. After 3 days clusters ofneuron-like cells began to appear in the LIF treated cultures, but notin control cultures. After 5 days the cultures were stained forneurofilament and neurons were counted (FIG. 4A), showing that therewere approximately 100 fold more neurons in the LIF treated culturesthan in controls. Neurons were also present in cultures treated withnerve growth factor (NGF), but there were only about 10% of those seenin the LIF treated cultures after 5 days. Experiments on DRG cellsisolated later in development (E15, E19, P2), showed a high proportion(80-100%) of neurons survived after 2 days in the presence of LIF (FIG.4A).

Limited dilution experiments indicate that LIF acts directly on theneurons, as the rate of survival is not influenced by cell number (FIG.4B). In addition, a LIF titration on the P2 DRG showed maximal activityover 10² U/ml and 50% activity at approximately 1.5 U/ml (FIG. 4C) whichis comparable to that observed with other neurotrophic factors (4,5,6).

These results indicate that LIF can act throughout embryonic sensoryneuron development in vitro. In neural crest cultures, it may act tostimulate neuronal differentiation and/or survival of the sensoryprecursors. Consistent with this, a subpopulation of neural crest cellswas found to specifically bind. ¹²⁵I-LIF, indicating that they have LIFreceptors. Others have implicated brain derived neurotrophic factor(BDNF) in the survival and/or differentiation of developing DRG cells.One possibility is that LIF, which is produced by mesoderm derived cellsin vitro, may be produced in peripheral tissue in vivo and act inconcert with the central nervous system derived BDNF in the developmentof the DRG.

The actions of LIF on the older DRG cultures show it to be aneurotrophic factor for sensory neurons in vitro like NGF. LIF acts as asurvival agent for postnatal and embryonic sensory neurons. The resultsherein indicate that LIF acts not only during the critical period oftarget innervation of the neurons but later as well. Thus, LIF may beexerting its effects throughout the development of sensory neurons andinto adulthood.

EXAMPLE 3 Effect of LIF on Spinal Cord Neurons

1. LIF Stimulates Process Outgrowth from Embryonic Spinal Cord

In Example 2, it was shown that LIF stimulates the development ofsensory neurons in cultures of neural crest obtained from E9 miceembryos. In these cultures, the crest cells migrate out from theembryonic spinal cord onto the fibronectin substratum and the sensoryneurons in the LIF cultures appear as clusters surrounding and at somedistance from the spinal cord explant. It had been noted that LIF alsoinfluenced the appearance of the spinal cord where the explant had beenleft in the cultures increasing their apparent viability and processoutgrowth. These experiments were repeated on spinal cord explants fromE10 embryos, where most of the neural crest has already migrated awayfrom the cord, but little neuronal differentiation occurred. In order tosee if LIF might be acting on neurons or their precursors in the spinalcord, the serum was removed from our assays to slow down glialproliferation without necessarily affecting neuronal differentiation. Asexpected, in these cultures there was very little cell migration awayfrom the explants, but there was still a great deal of process outgrowthin the LIF treated cultures (FIG. 5). The processes extended straightout from the explants, some in bundles and some as single processes,onto the substratum. There was also a limited degree of arborization ofthe processes. The stimulation of process outgrowth first becameapparent at day 3 and increased up to a maximum at day 7.

These observations indicate that LIF may contribute to the processoutgrowth and development of spinal cord neurons. To further test this,single cell suspensions of spinal cord cells were made and plated in thepresence and absence of LIF to see if the effect could be observed indissociated cultures. The advantage of these cultures is that an exactnumber of cells can be plated in each well as opposed to explants ofdifferent sizes and thus it may be easier to quantitate the effect ofLIF.

When these cells were plated at fairly high cell density in both 96 welland HLA plates, they spontaneously aggregated into discrete clusters andprocesses emanated from these clusters and appeared to form bridges withother clusters (FIG. 6). That these processes were definitely ofneuronal origin was established by staining the cultures forneurofilament. All the processes in both LIF treated and controlcultures stained positively with the anti-neurofilament antibody (FIG.6). In the presence of LIF far more of these processes were present thanin controls (FIG. 6). Almost all of the cell clusters in the LIFcultures emanated processes whereas most of the clusters in the controlshad no processes. Further, there were generally more processes percluster in the LIF cultures. This effect was observed by day 2 and wasmost obvious at day 5, by which time the number of processes in thecontrol cultures had begun to diminish. At this time, the average numberof processes in the LIF treated cultures was approximately 10 times thatin the controls (FIG. 7).

EXAMPLE 4

LIF Stimulates an Increase in the Number of Neurons in Spinal CordCultures.

One possibility to account for the stimulation of process outgrowth byLIF is that it stimulates the survival of precursors and/ordifferentiation of neurons in the spinal cord cultures. Initially, thetotal number of cells present in the cell cultures in the presence andabsence of LIF was investigated. Cell counts were performed from 96 wellplates after 3 and 5 days in vitro. As shown in FIG. 13, there was asmall increase in total cell numbers in the presence of LIF. These dataalso show that there was little increase in cell number in either LIF orcontrol culture, suggesting that little proliferation has occurred.

The increase in numbers in the LIF cultures might either be a smallsurvival effect or an affect on a subpopulation of cells within theculture, i.e. the neurons. However, this method of analysis does notallow for the identification of neurons in the population. To determineif there were a significant effect on neuron number, as opposed to theentire population of cells which developed in the culture system, theE10 cells were plated at low density onto irradiated Balb/c-3T3monolayers. Under these conditions, the cultures could be stained forneurofilament and individual neurons counted. By day 4 there wereapproximately 2 fold more neurons in the LIF treated cultures. Inculture where 10,000 spinal cord cells were plated, 1920 neurons wereobserved in the LIF treated cultures compared to 998 in controls. Incultures where 2500 spinal cord cells were plated, there were 625neurons in the LIF treated cultures compared to 343 in controls. By day7 of culture there was still good survival of neurons in the LIFcultures, whereas almost all of the neurons had died in the controlcultures. These data suggest that LIF stimulates both thedifferentiation and survival of spinal cord neurons.

These experiments show that LIF stimulates process outgrowth from theundifferentiated trunk neural tube and from the embryonic spinal cord.Thus, LIF appears to be acting to stimulate the differentiation ofspinal neurons which innervate the peripheral tissues of the body. Thethree major classes of neurons which do this are the lower motor neuronsof the spinal cord and the preganglionic sympathetic and parasympatheticchains. As it is not yet possible to distinguish which of these classesLIF may be effecting, it can be speculated that the lower motor neuronswould be good candidates given that the processes emanating from thetube are thick and extend long distances from the neural tube. Onlylower motor neurons do this in vivo from the spinal cord. In addition,LIF has been found in the muscle which is the natural target of lowermotor neuron innervation. A cohesive hypothesis is that LIF is themuscle derived target factor for these motor neurons. It stimulates themto extend processes toward the target and then acts as a survival factorfor the neurons which have successfully innervated the muscle.

EXAMPLE 5 Binding and Retrograde Labelling Experiments

Example 2 shows that LIF supports the survival of the majority ofsensory neurons form newborn dorsal root ganglia. This is evident evenat very low cell numbers—single neurons could be supported—indicatingthat LIF probably acts directly on neurons and not via an accessorycell. To formally prove that sensory neurons express high affinity LIFreceptors, binding studies on isolated sensory neurons were carried outin vitro. As shown in FIG. 8, greater than 50% of cells identified asneurons by their expression of neurofilament bound significant amountsof ¹²⁵I-LIF, all of which was inhibited by the addition of cold LIF.Furthermore, there was negligible cold-inhibitable binding of ¹²⁵I-LIFto non-neuronal cells in the culture.

These results show that mature sensory neurons do express high affinityreceptors for LIF and that the accessory cells, such as Schwann cells,do not. This strongly argues for the direct neuronal action of LIF,which was predicted from the limiting dilution studies (Example 2) inwhich LIF supported the survival of very low numbers of sensory neurons.Studies with radiolabelled NGF have shown that both Schwann cells andneurons bind NGF in vitro, although it is not clear whether thisreflects the steady—state in vivo situation. Apart from LIF, no otherfactors have been shown binding limited to the neuronal component.

The observed distribution of receptors fits well with results on invitro survival that show that the vast majority of sensory neuronssurvive in the presence of LIF. The restricted distribution alsosuggests that LIF receptors may be limited to the neuronal lineageduring sensory ganglia development.

Having demonstrated the presence of LIF receptors on sensory neurons invitro, it was next investigated whether receptor mediated uptake of LIFwould result in retrograde transport to the sensory neuron soma.Experiments using nerve ligation were carried out to determine if therewas any retrograde transport of ¹²⁵I-LIF by neurons with axons in thesciatic nerve. It was found that there was significant accumulation ofradioactivity in the distal segment of the nerve after injection of¹²⁵I-LIF, into both the foot or leg (see Table 1). The time course ofthis accumulation suggested that it was due to retrograde transport andnot to other mechanisms; furthermore there was no evidence of the distalaccumulation of ¹²⁵I-FGF after injection.

In order to examine more closely which neurons were involved in theretrograde transport of LIF, adult mice were again injected in the skinor muscle, but this time with the sciatic nerve intact. In those animalsinjected in the skin of the foot, after 16 hours there was a significantaccumulation of radioactivity in the sensory ganglia centered on lumbarganglion 4 (L4; FIG. 9). There was a very much smaller accumulation ofradioactivity in those animals injected in the muscle and this appearedto be more rostral (FIG. 9). Although FGF has been shown to support arange of neurons, including sensory neurons, there was no evidence ofaccumulation of ¹²⁵I-FGF in the lumbar DRG or spinal cord. TABLE 1Injection of LIF into adult mice with ligated sciatic nerve Accumulationof LIF in nerve uM/2 mm Time after injection Proximal stump Distal stumpInjection into the footpad  7 hrs 0.144 ± .024 0.349 ± .084 16 hrs 0.170± .034 0.777 ± .108 24 hrs 0.060 ± .006 0.551 ± .045 Injection into thegastrocnemus muscle  7 hrs 0.109 ± .008 0.488 ± .128 16 hrs 0.137 ± .0140.550 ± .135 24 hrs 0.069 ± .004 0.399 ± .138

The sciatic nerve was ligated in the mid high region of the adult miceand 1 μCi of ¹²⁵I-LIF was injected either into the footpad of calf.After the various times the nerve was removed and 2 mm sections eitherside of the ligature were taken and radioactively measured in a gammacounter.

In newborn mice there was a greater accumulation of radioactivity forboth the leg and foot injections. The skin injection again was centeredon L4 (FIG. 10). The transport from the muscle injection was morewidespread and may reflect the greater spread from the injection site inthese small animals (FIG. 10). Again in both cases the accumulation ofradioactivity in the L4 ganglia followed a time course consistent withretrograde transport.

Autoradiographic examination of histological sections through L4 gangliafrom both adult and newborn animals injected with ¹²⁵I-LIF into thefootpad has revealed the presence of radioactive material in asubpopulation of neurons (FIG. 11). The number of neurons withsignificant number of grains is between 5-10% of the population (FIG.12), again there is no evidence of radioactivity associated withnon-neuronal cells (FIG. 11).

A major finding in accordance with this aspect of the present inventionis that LIF is retrogradely transported in a manner resembling NGF. Thisre-enforces the view that the expression of LIF receptors is not an invitro artefact and more importantly implicates LIF as a neurotrophicmolecule for sensory neurons. As far as the present inventors know thisis the only neurotrophic molecule, outside of NGF, that has been shownto be transported in such a manner, although there is evidence that FGFcan be transported antero gradely in retinal ganglion cells. LIF, likeNGF, does not appear to be transported antero gradely as there is noevidence of accumulation of the molecule in the spinal cord. It appearsthat LIF is not transported by motor neurons in the sciatic nerve nordoes it appear to be transported in the sympathetic or parasympatheticnervous systems. This probably indicates that LIF is also capable ofexerting a biological effect on the nervous system by directly bindingto the cell surface and not undergoing receptor mediated transport. Thiswould appear to be the primary mode of action of LIF on a wide varietyof cells which include muscle, platelets, embryonal stem cells and somehaemopoietic cell lines.

Although retrograde transport of NGF seems to, be required for some ofits biological action, no such evidence exists for LIF. The similaritiesof action of the two factors in the developing sensory neurons suggeststhat this process may be necessary to deliver a sufficient biologicalsignal from the periphery to the cell soma. Such a suggestion appearstoo simplistic given the findings that NGF injected into the cell somadoes not result in neuron survival. This suggests that it is thereceptor-ligand complex that is important in signal delivery.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

REFERENCES

-   1. Le Dourin, N. M. Science 231: 1515-1522, 1986.-   2. Ziller, C., Fauquet, M., Kalcheim, C., Smith, J., & Le    Douarin, N. M. Dev. Biol. 120: 101-111, 1987.-   3. Anderson, D. J. Neuron 3: 1-12, 1989.-   4. Levi-Montalcini, R. Annu. Rev. Neurosci. 5: 341-362, 1982.-   5. Barde, Y. Neuron. 2: 1525-1534, 1989.-   6. Barbin, G., Manthorpe, M., & Varon, S. J. Neurochem. 43:    1468-1478, 1984.-   7. Murphy, M., Drago, J. & Bartlett, P. J. Neurosci. Res. 25:    463-475, 1990.-   8. Nissl, F. Allg. Z. Psychiat. 48: 197-198, 1982.-   9. Good, M. F., Boyd, A. W. & Nossal, G. J. V. J. Immunol. 130:    2046-2055, 1983.-   10. Eckenstein, F. P., Baughman, R. W. & Quinn, J. Neurosci. 25:    457-474, 1988.-   11. Example 1, isolation of spinal cord cells.-   12. Hilton et al., 1990.-   13. Hilton, D. J., Nicola, N. A., & Metcalf, D. Anal. Biochem. 173:    359-367, 1988.-   14. Shaw, G., Osborne, M., * Weber, H. Eur. J. Cell. Biol. 26:    68-82, 1981.-   15. Ju, G., Hokfelt, T., Brodin, E., Fahrenkrug, J., Fisher, J. A.,    Frey, P., Elde, R. P., & Brown, J. C. Cell Tiss. Res. 247: 417-431,    1987.-   16. Gibbins, I. L., Furness, J. B., & Costa, M. Cell Tiss. Res. 248:    417-437, 1987.-   17. Juurlink, B. H. J., Munoz, D. G., & Devon, R. M. J. Neurosci.    Res. 26: 238-241, 1990.-   18. Kessler, J. A., & Black, I. B. Proc. Natl. Acad. Sci. USA 77:    649-652, 1980.-   19. Lawson, S. N., Caddy, K. W. T., & Biscoe, T. J. Cell Tiss. Res.    153: 399-413, 1974.-   20. Malcheim, C., & Jandreau, M. Dev. Brain Res. 41: 79-86, 1988.-   21. Malcheim, C., Barde, Y. A., Thoenen, H., & Le Douarin, N. M.    EMBO J. 6: 2871-2873, 1987.

1. A method for regulating neuron development and/or regeneration and/ormaintenance in a mammal comprising administering to said mammal aneffective amount of leukaemia inhibitory factor (LIF) for a time andunder conditions sufficient to permit the development of neuralprecursor cells into neurons and/or to promote survival of said neuralprecursor cells.
 2. The method according to claim 1 wherein the neuronsare located in the peripheral nervous system.
 3. The method according toclaim 1 wherein the neurons are located in the central nervous system.4. The method according to any one of the preceeding claims wherein themammal is a human.
 5. The method according to claim 4 wherein the routeof administration is by intravenous or intramuscular injection orinfusion or by gene therapy or by retrograde labelling.
 6. The methodaccording to claim 5 wherein the LIF is mammalian LIF.
 7. The methodaccording to claim 6 wherein the mammalian LIF is mouse, rat, human orlivestock animal LIF.
 8. The method according to claim 7 wherein themammalian origin of LIF and the mammal to be treated belong to the samespecies.
 9. The method according to any one of the preceding claimswherein the effective amount of LIF is from about 0.01 to about 10,000μg/kg body weight.
 10. The method according to claim 9 furthercomprising the simultaneous or sequential administration of one or moreother neuron stimulating factors.
 11. The method according to claim 10wherein the other neuron stimulating factors comprise FGF, CNTF, NGFand/or BNDF and/or one or more neurotrophic factors.
 12. The methodaccording to claim 11 wherein each other neuron stimulating factor isadministered in an effective amount of from about 0.01 to about 10,000μg/kg body weight.
 13. The method according to claim 2 wherein theneurons are sensory neurons.
 14. The method according to claim 1 or 3wherein the neurons are spinal cord neurons.
 15. A method for regulatingspinal cord development and/or repair and/or maintenance and/orregeneration in a mammal comprising administering to said mammal aneffective amount of LIF for a time and under conditions sufficient or toincrease the number, to maintain or regenerate spinal cord neuronsand/or neurite processes.
 16. The method according to claim 15 whereinthe mammal is a human.
 17. The method according to claim 15 or 16wherein the route of administration is by intravenous or intramuscularinjection or infusion or by gene therapy or by retrograde labelling. 18.The method according to claim 17 wherein the LIF is mammalian LIF. 19.The method according to claim 18 wherein the mammalian LIF is mouse,rat, human or livestock animal LIF.
 20. The method according to claim 19wherein the mammalian origin of LIF and the mammal to be treated belongto the same species.
 21. The method according to any one of claims 15 to20 wherein the effective amount of LIF is from about 0.01 to about10,000 μg/kg body weight.
 22. The method according to claim 21 furthercomprising the simultaneous or sequential administration of one or moreother neuron stimulating factors.
 23. The method according to claim 22wherein the other neuron stimulating factors comprise FGF, CNTF, NGFand/or BNDF and/or one or more neurotrophic factors.
 24. The methodaccording to claim 23 wherein each other neuron stimulating factor isadministered in an effective amount of from about 0.01 to about 10,000μg/kg body weight.
 25. A method of treating a disease and/or injury to anervous system in a mammal comprising administering to said mammal aneffective amount of LIF for a time and under conditions sufficient toameliorate the disease and/or injury.
 26. The method according to claim25 wherein the mammal is a human.
 27. The method according to claim 26wherein the nervous system is the peripheral nervous system.
 28. Themethod according to claim 26 wherein the nervous system is the centralnervous system.
 29. The method according to claim 27 or 28 wherein thedisease or injury is Cerebral Palsy, trauma induced paralysis, vascularischaemia associated with stroke, neuronal tumours, motorneuron disease,Alzheimer's disease, multiple sclerosis, Parkinson's disease,Huntington's disease, peripheral neuropathies associated with diabetes,heavy metal or alcohol toxicity, renal failure and/or infectiousdisease.
 30. The method according to claim 29 wherein the infectiousdiseases comprise herpes, rubella, measles, chicken pox, HIV and/orHTLV-1.
 31. The method according to any one of claims 25 to 30 whereinthe LIF is mammalian LIF.
 32. The method according to claim 31 whereinthe mammalian LIF is mouse, rat, human or livestock animal LIF.
 33. Themethod according to claim 32 wherein the mammalian origin of LIF and themammal to be treated belong to the same species.
 34. The methodaccording to claim 26 or 30 wherein the effective amount of LIF is fromabout 0.01 to about 10,000 μg/kg body weight.
 35. The method accordingto claim 34 further comprising the simultaneous or sequentialadministration of one or more other neuron stimulating factors.
 36. Themethod according to claim 35 wherein the other neuron stimulatingfactors comprise FGF, CNDF, NGF and/or BNDF and/or one or more otherneurotrophic factors.
 37. A pharmaceutical composition comprising LIF,one or more other neuron stimulating factors and one or morepharmaceutically acceptable carriers and/or diluents.
 38. Thepharmaceutical composition according to claim 37 wherein the neuronstimulating factors comprise FGF, CNDF and/or BNDF and/or one or moreother neurotrophic factors.
 39. The use of LIF in the manufacture of amedicament for the regulation of neurons and/or treatment of diseases orinjury to a nervous system in a mammal.
 40. The use according to claim39 wherein the nervous system is the peripheral nervous system, thecentral nervous system and/or the spinal cord.
 41. The use according toclaim 39 or 40 further comprising the use of one or more other neuronstimulating factors.
 42. The use according to claim 41 wherein the otherneuron stimulating factors comprise FGF, CNTF and/or BNDF and/or one ormore other neurotrophic factors.